INTRODUÇÃO: A inflamação das vias aéreas na asma envolve respostas imunes inatas. Os receptores do tipo Toll (Toll-like receptors, TLRs) e a citocina linfopoetina do estroma tímico (thymic stromal lymphopoietin, TSLP) estão envolvidos na inflamação brônquica da asma, mas a expressão destas proteínas em vias aéreas grandes e pequenas de asmáticos ainda não foi investigada. Os objetivos deste estudo foram analisar a expressão protéica de TLR2, TLR3, TLR4 e TSLP em vias aéreas grandes e pequenas de asmáticos, comparar sua expressão entre asmáticos tabagistas e não tabagistas e investigar se a expressão dos TLRs está associada à infecção por Chlamydophila pneumoniae e Mycoplasma pneumoniae. MÉTODOS: Foram analisadas por método imuno-histoquímico e análise de imagens as expressões de TLR2, TLR3, TLR4 e TSLP em vias aéreas grandes e pequenas de 24 indivíduos falecidos por asma (13 não tabagistas e 11 tabagistas) e 9 controles não asmáticos. A análise das proteínas foi realizada em quatro regiões das vias aéreas: camadas epitelial, interna, muscular e externa. A presença de C. pneumoniae e M. pneumoniae no tecido pulmonar foi investigada por meio de reação em cadeia da polimerase em tempo real. RESULTADOS: Os indivíduos asmáticos apresentaram maior expressão de TLR2 nas camadas epitelial e externa de vias aéreas grandes e pequenas, e maior TLR2 na camada muscular de vias aéreas pequenas. Asmáticos tabagistas tiveram menor expressão de TLR2 nas camadas interna e externa de vias aéreas pequenas do que asmáticos não tabagistas. Indivíduos asmáticos tiveram maior expressão de TSLP na camada epitelial e externa de vias aéreas grandes, aumento de TLR3 na camada externa de vias aéreas grandes e aumento de TLR4 na camada externa de vias aéreas pequenas. O DNA de C. pneumoniae e M. pneumoniae não foi detectado em nenhum indivíduo asmático ou controle. CONCLUSÕES: Os receptores da imunidade inata TLR2, 3 e 4 e a citocina TSLP estão aumentados nas vias aéreas de pacientes falecidos por asma, e a expressão dos TLRs não está associada à presença de Chlamydophila pneumoniae e Mycoplasma pneumoniae nos pulmões. O tabagismo em asmáticos parece reduzir a expressão de TLR2 em vias aéreas pequenas. Estes resultados sugerem que os TLRs 2, 3 e 4 e a TSLP podem contribuir com a inflamação brônquica presente em exacerbações graves de asma e que as bactérias C. pneumoniae e M. pneumoniae não estão envolvidas em óbitos por asma / INTRODUCTION: Airway inflammation in asthma involves innate immune responses. Toll-like receptors (TLRs) and the cytokine thymic stromal lymphopoietin (TSLP) are involved in bronchial inflammation in asthma, but the expression of these proteins in large and small airways of asthmatics has not been investigated. The aims of this study were to analyze the protein expression of TLR2, TLR3, TLR4 and TSLP in large and small airways of asthmatics, to compare their expression in smoking and nonsmoking asthmatics and to investigate if TLR expression in associated with infection by Chlamydophila pneumoniae and Mycoplasma pneumoniae. METHODS: Using immunohistochemistry and image analysis, we investigated the expression of TLR2, TLR3, TLR4 and TSLP in large and small airways of 24 fatal asthma patients (13 nonsmokers and 11 smokers) and 9 nonasthmatic controls. The protein expression was analyzed in four regions of the airways: epithelial, internal, airway smooth muscle and outer layers. C. pneumoniae and M. pneumoniae presence in lung tissue was analyzed by real-time polymerase chain reaction. RESULTS: Fatal asthma patients had increased expression of TLR2 in the epithelial and outer layers of large and small airways, and also higher TLR2 in the muscle layer of small airways. Smoking asthmatics had lower TLR2 in the inner and outer layers of small airways than nonsmoking asthmatics. TSLP was increased in the epithelial and outer layers of large airways. Asthmatics also had greater TLR3 in the outer layer of large airways and greater TLR4 in the outer layer of small airways. C. pneumoniae and M. pneumoniae DNA was not detected in asthmatics or controls. CONCLUSIONS: Innate immunity receptors TLR2, 3 and 4 and innate cytokine TSLP are increased in the airways of fatal asthma patients, and TLRs expression is not associated with the presence of Mycoplasma pneumoniae and Chlamydophila pneumoniae in the lungs. Smoking may reduce TLR2 expression in the small airways of asthmatics. These results suggest that TLR2, 3, 4 and TSLP may contribute to the bronchial inflammation seen in severe exacerbations of asthma and that M. pneumoniae and C. pneumoniae are not involved in fatal asthma exacerbations

Hepatitis B virus (HBV) infection is one of the major causes of chronic and cancerous liver disease. Elimination of HBV from chronically infected patients by recombinant interferon α (IFNα) monotherapy shows that the mechanisms of the innate immunity play an important role in suppressing viral infection. However, the mechanisms of recognition of the HBV genome and its escape from the mechanisms of natural immunity are still little known. One of the principal factors enabling the virus to escape from cellular restriction mechanisms is the HBx viral protein. HBx is a 154 amino acid pleiotropic multifunctional protein affecting transcription, signal transduction, cell cycle, protein degradation, apoptosis, and chromosomal stability in the host cell. Previous results from our laboratory have shown that activation of the MEK1/2-ERK signaling pathway in plasmacytoid dendritic cells leads to inhibition of IFNα production. The aim of my work was to determine whether HBx activates the MEK1/2-ERK pathway and thus inhibits IFN type I production also in hepatocytes. For this purpose, I monitored HBx production in the Huh7 hepatoma cell line by transfecting the bicistronic plasmid pHBx- IRES-EGFP and Western blotting. Using the same method, I monitored activation of the MEK1/2-ERK signaling pathway by ERK...

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Invariant natural killer T (iNKT) cells are a versatile subclass of T lymphocytes which recognize glycolipid antigens. iNKT cells are capable of rapidly producing a broad array of cytokines in response to stimulation; thus, they play an important role in the early regulation of a variety of immune responses. It was hypothesized that iNKT cells express functional Toll-like receptors (TLRs) and that stimulation of TLRs by their ligands modulates iNKT cells responses. In the first objective, it was revealed that upon stimulation with anti-CD3 monoclonal antibody and interferon (IFN)-α, expression of TLRs was enhanced in iNKT cells. Furthermore, stimulation of iNKT cells with TLR ligands led to a significant increase in the expression of several cytokines. In the second objective, the mechanisms behind the modulatory effects of the TLR9 ligand (CpG-ODN) on iNKT cells were determined. Altogether, these findings suggest a direct role for TLRs in iNKT cell activation. / Ontario Graduate Scholarship

Immunotherapy became a very promising approach for cancer therapy. Tumor cells are eliminated using the body's own immune system with minimal negative effect on healthy tissue. This thesis is focused on immunotherapy based on activation of innate immunity, specifically on intratumoral application of ligands stimulating phagocytosis and Toll-like receptor ligands. This therapeutic approach was tested in several types of tumor mouse models, such as melanoma B16-F10, pancreatic adenocarcinoma, and pheochromocytoma. The composition of the therapeutic mixture as well as the application schedule were optimized in our studies. Subsequently the underlying mechanisms involved in tumor elimination during this therapy were investigated.

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Multiple Sclerosis (MS) is a Central Nervous System disease, mediated by the Immune System, whose symptoms occur in episodes of relapses. Interferon-beta (IFN-β) is considered a safe treatment for the reduction of relapses, but its mechanisms of action have not yet been clear. Studies have shown involvement of tumor necrosis factor alpha (TNF-α) and of Interleukin-10 (IL-10) in the immunopathogenesis of MS. The role of IL-32, a proinflammatory cytokine has role on several chronic inflammatory diseases, was not elucidated in MS. The effect of IFN-β on these cytokines and disease severity, as measured by the Expanded Disability Status Scale (EDSS), has not yet been established. Objective. The objective of the present study was to evaluate TNF-α, IL-10 and IL-32γ concentrations in the peripheral blood and gene expression of patients with IFN-β. Methods. The sample were patients of the Department of Neurology of the Clinics Hospital of the Federal University, and healthy individuals. Blood collection, blood culture with lipopolysaccharide (LPS), Toll 4 receptor agonist (TLR4), and PAM3Cys, TLR2 agonist, and the quantification of cytokines by real-time polymerase chain reaction were performed. Mann Whitney tests were used for statistical analysis of unpaired data, Wilcoxon for paired samples and Spearman's correlation test, adopting significance level p <0.05. Results. Of the 30 MS patients, 19 were treated with IFN-β and 11, untreated, with a mean age of 40.52 and 42 years, respectively, and female prevalence. TNF-α did not differ between groups but it was less produced after stimulation with Pam3Cys in treated patients compared to controls and untreated patients. IL- 10 concentrations were higher in cultures with LPS in patients treated compared to healthy controls. The mean EDSS of patients treated with IFN-β and untreated did not differ, and the correlation between and TNF-α and IL-10 concentrations produced in blood cultures and EDSS was not significant in the patients. There was a significant correlation between TNF-α concentrations and disease time in untreated patients in non-stimulated cultures and those with TLR2 agonist stimulus. Gene expression of IL-32γ was higher in IFN-β treated patients compared to controls. The gene expression of cytokines correlated positively and significantly in patients and controls and the IL-10 expression was correlated negative e significantly with the disease time in untreated patients. Conclusions. IFN-β reduced patients' response to Pam3Cys. IL-10 was higher in treated patients relative to controls. The correlations were not conclusive about the possible association between these cytokines and the clinic parameters of the disease. IL-32γ was higher in patients treated with IFN-β than in healthy subjects. / Introdução. A Esclerose Múltipla (EM) é uma doença do Sistema Nervoso Central, mediada pelo Sistema Imune,cujos sintomas ocorremem episódios de surtos.OInterferon-beta (IFN-β) é considerado um tratamento seguro para redução dos surtos, masseus mecanismos de ação ainda não foram bemesclarecidos. Estudos mostraram participaçãodo fator de necrose tumoral alfa (TNF-α) e da Interleucina-10 (IL-10) na imunopatogênese da EM. O papel da IL-32, citocina pró-inflamatória atuante emvárias doenças inflamatórias crônicas, não foi elucidado na EM. O efeito do IFN-β nestas citocinas e na gravidade da doença, medida pela Escala do Estado de Incapacidade Expandida (EDSS), ainda não foi estabelecido. Objetivo.O objetivo do presente estudo foi avaliar as concentrações de TNF-α, IL- 10 e IL-32γ no sangue periférico de pacientes com EM tratados com IFN-β e não tratados. Métodos.Foram recrutados portadores da doença, no Serviço de Neurologia do Hospital das Clínicas da Universidade Federal,e indivíduos sadios. Foi feita a coleta de sangue, hemoculturas com lipopolissacarídeo (LPS), agonista do receptor do tipo Toll 4 (TLR4), e PAM3Cys, agonista de TLR2, e a quantificação gênica das citocinas por reação em cadeia da polimerase em tempo real. Foram utilizados os testes Mann Whitney, para análise estatísticados dados não pareados, Wilcoxon para as amostras pareadas e o teste de correlação de Spearman, adotando nível de significância p<0,05. Resultados. Dos 30 pacientes com EM, 19 eram tratados com IFN-β e 11, não tratados, com idade média de 40,52 e 42 anos, anos, respectivamente, e prevalência do sexo feminino. As concentrações de IL-10 foram mais elevadasnas culturas dos pacientes tratados com IFN-β estimuladas com LPS comparado aos controles sadios.TNF-α não diferiu entre os grupos, mas foi menos produzido após estimulação com Pam3Cys nos pacientes tratados comparado aos controles e aos pacientes não tratados. O EDSS médio dos pacientes tratados com IFN-β e dos não tratados não diferiu, e a correlação entre e as concentrações de TNFα e IL-10 produzidas nas hemoculturas e o EDSS não foi significante nos pacientes. Houve correlação significante entre as concentrações de TNF-α e o tempo de doença nos pacientes não tratados nas culturas não estimuladas e naquelas com estímulo de agonista de TLR2. A expressão gênica de IL-32γ foi mais elevada nos pacientes tratados com IFN-β comparado aos controles. As expressões gênicas das citocinas se correlacionaram positiva e significantemente nos pacientes e controles e a expressão de IL-10 se correlacionou negativa e significantemente com o tempo de doença nos pacientes não tratados. Conclusões. O IFN-β reduziu a resposta dos pacientes ao Pam3Cys. IL-10 foi mais elevada nos pacientes tratados em relação aos controles. As correlações não foram conclusivas sobre a possível associação entre essas citocinas e os parâmetros clínicos da doença. IL-32γ foi mais elevada nos pacientes tratados com IFN-β em relação às pessoas sadias.

O objetivo deste trabalho foi caracterizar a formação e progressão de lesões periapicais induzidas experimentalmente em dentes de camundongos knockout para receptores toll-like 2 (TLR2 KO) comparados a animais wild-type (WT). As lesões periapicais foram induzidas nos primeiros molares inferiores de 28 camundongos WT e de 27 camundongos TLR2 KO. Decorridos 7, 21 e 42 dias da indução da lesão periapical, os animais foram submetidos à eutanásia em câmara de CO2, as mandíbulas foram removidas e submetidas ao processamento histotécnico. A seguir, cortes representativos foram corados com hematoxilina e eosina (HE), para descrição do tecido pulpar e das regiões apical e periapical, em microscopia óptica convencional, e mensuração da área das lesões periapicais, em microscopia de fluorescência. Espécimes sequenciais foram avaliados por meio de: histoenzimologia para a atividade da TRAP, para identificação de osteoclastos; coloração de Brown & Brenn, para localização de bactérias; e imunoistoquímica, para identificação de marcadores da osteoclastogênese (RANK, RANKL, OPG). Os resultados numéricos obtidos da análise morfométrica da extensão da área das lesões periapicais e do número de osteoclastos foram submetidos à análise estatística por meio dos testes não-paramétricos de Mann-Whitney e Kruskal-Wallis, utilizando o software SAS (Statistical Analysis System) for Windows versão 9.1.3. O nível de significância adotado foi de 5%. Os resultados da coloração de Brown & Brenn e Imunoistoquímica foram expressos de maneira qualitativa. O grupo de animais WT apresentou diferença significante na extensão da área das lesões periapicais e no número de osteoclastos entre os períodos experimentais de 7 e 42 dias (p<0,05) e entre 21 e 42 dias (p<0.05). Por outro lado, no grupo de animais TLR2 KO, as diferenças para a extensão da área das lesões periapicais e número de osteoclastos foram encontradas entre os períodos experimentais de 7 e 21 dias (p<0,05) e entre 7 e 42 dias (p<0,05). Quando os períodos dos grupos foram comparados entre si, foram encontradas diferenças estatísticas entre todos os períodos experimentais, tanto para a análise morfométrica da extensão da área das lesões periapicais, quanto para o número de ostoclastos (p<0,05). A análise descritiva do tecido pulpar e das regiões apical e periapical, por meio da coloração de HE, bem como da localização das bactérias, por meio da coloração de Brown & Brenn, não mostrou diferenças entre os dois grupos de animais. Com relação à Imunoistoquímica, as marcações foram semelhantes entre os dois grupos de animais, exceto para as marcações de RANK, as quais não foram encontradas nas lesões periapicais do grupo de animais TLR2 KO. A partir das metodologias empregadas e dos resultados obtidos pode-se concluir que na ausência do TLR2, os animais desenvolveram lesões periapicais significantemente maiores (com maior presença de osteoclastos) quando comparados aos animais WT, sugerindo o importante papel desse receptor na resposta imune e inflamatória do organismo no sentido de combater a infecção do sistema de canais radiculares e dos tecidos perirradiculares. / The aim of the present study was to characterize the formation and progression of periapical lesions experimentally induced in the teeth of toll-like receptors 2 knockout (TLR2 KO) mice compared to wild-type (WT) mice. Periapical lesions were induced in the lower first molars of 28 WT and 27 TLR2 KO mice. After 7, 21 and 42 of periapical lesion induction, the animals were euthanized in a CO2 chamber, and the mandibles were removed and subjected to histotechnical processing. Representative histological sections were stained with hematoxylin and eosin (HE) for description of the features of the pulp tissue and the apical and periapical regions under conventional optical microscopy, and for determination of the size of the periapical lesions under fluorescence microscopy. Sequential specimens were evaluated by: TRAP histo-enzymology for identification de osteoclasts; Brown & Brenn staining for localization of bacteria; and immunohistochemistry for identification of osteoclastogenesis markers (RANK, RANKL, OPG). Data from the morphometric evaluation of the size of periapical lesions and the number of osteoclasts were subjected to statistical analysis by the nonparametric Mann-Whitney and Kruskal-Wallis tests, using the SAS (Statistical Analysis System) software for Windows version 9.1.3. A significance level of 5% was set for all analyses. Data from the Brown & Brenn staining and immunohistochemical analysis were displayed qualitatively. The group of WT mice presented statistically significant difference in the periapical lesion size and number of osteoclasts between the 7- and 42-day experimental periods (p<0.05) as well as between 21 and 42 days (p<0.05). On the other hand, in the group of TLR2 KO mice, significant differences in the periapical lesion size and number of osteoclasts were found between the 7- and 21-day experimental periods (p<0.05) as well as between 7 and 42 days (p<0.05). Comparison of the periods within each group revealed statistically significant differences among all experimental periods for the morphometric evaluation of the size of the periapical lesions and number of osteoclasts (p<0.05). Descriptive analysis of pulp tissue and apical and periapical regions by HE staining and localization of bacteria by Brown & Brenn staining did not show significant differences between the two groups of animals. The immunohistochemical results showed similar immunostaining in both groups of animals, except for RANK expression, which was not observed in the periapical lesions of the TLR2 KO mice. Based on the employed methodology and the obtained results it may be concluded that in the silence of TLR2, the animals developed superior size of periapical lesions (with higher presence of osteoclasts) compared to WT animals, suggesting the important role of this receptor during the immune and inflammatory response against the infection of root canal system and periapical tissues.

Durante uma infecção o número de linfócitos T aumenta dramaticamente. A fase de expansão clonal é seguida pela redução do nível de células T ativadas que depende, em parte, de um processo de morte celular induzida por ativação (AICD). Nosso grupo mostrou que APCs quando são estimuladas com LPS, um agonista de TLR4, produzem PGE2, que inibe a AICD de linfócitos T CD4+ através da supressão da expressão de CD95L (Weinlich et al, 2008). Porém, os efeitos da estimulação direta de TLR nos linfócitos T sobre o processo de AICD foram pouco elucidados. Sendo assim, o projeto tem como objetivo verificar se a estimulação dos diversos TLRs com seus agonistas é capaz de modular a AICD de hibridomas de linfócito T DO11.10. Inicialmente, demonstramos que este hibridoma expressa os TLR1, 2, 3, 4, 5, 6, 7, 9 e 11. Dentre todos os agonistas utilizados somente o Imiquimod, um agonista de TLR7, reduziu a morte celular quando adicionado durante a indução de AICD. O efeito protetor desse agonista foi através da inibição da expressão de CD95L, tanto no nível de mRNA quanto protéico. / During an infection the number of T lymphocytes increases dramatically. The clonal expansion phase is followed by reducing the level of activated T cells that depends, in part, a process of cell death induced by activation (AICD). Our group showed that when APCs are stimulated with LPS, a TLR4 agonist, produce PGE2, which inhibits the AICD of CD4 + T cells by suppressing the expression of CD95L (Weinlich et al, 2008). However, the effects of direct stimulation of TLR on T cells on the process of AICD were little explained. Therefore, the project aims to determine whether the stimulation of different TLRs to their agonist is able to modulate AICD of T lymphocyte hybridoma DO11.10. Initially, we demonstrated that this hybridoma expresses TLR1, 2, 3, 4, 5, 6, 7, 9 and 11. Among all agonists used only imiquimod, a TLR7 agonist, reduced cell death when added during the induction of AICD. The protective effect of this agonist was by inhibiting the expression of CD95L, both at the mRNA and protein.

A aterosclerose é um processo complexo, multifatorial que ainda não está totalmente esclarecido. Foi proposto que a resposta imune mediada por processos infecciosos e/ou inflamatórios influencia na patogênese de lesões ateroscleróticas. Os receptores TolI-likes (TLRs) estão envolvidos na resposta inata e em outros eventos fisiológicos através da interação com seus ligantes endógenos e exógenos e talvez envolvidos no processo aterogênico. Tem por objetivo analisar a expressão dos receptores Toll-like 2 e 4 (TLR2 e TLR4) associando o processo de sinalização com a presença de agentes infecciosos tais como a Chlamydia pneumoniae (CP) e Mycoplasma pneumoniae (MP), em pacientes com infarto do miocárdio (MI) e em aneurismas aórticos. Foram obtidos fragmentos de aortas ascendentes de pacientes submetidos à cirurgia de revascularização do miocárdio (G1, n=13) e de fragmentos de pacientes submetidos à cirurgia de correção de aneurisma aórtico (G2, n=14). Amostras congeladas e parafinadas foram analisadas por Imunohistoquímica (lHO) e Hibridização in situ (HIS) para detecção e localização da presença dos patógenos e TLRs. Realizou-se uma semiquantificação em microscópio (O, ausente; 1, discreto e focal; 2, moderado e focal e 3, intenso e difuso). Observou-se o grau de inflamação e de acúmulo de gordura. Outrossim, realizou-se PCR em tempo real (SYBR Green) para pesquisa de DNA de CP e MP, como também análise da expressão de mRNA de TLR2 e de TLR4. Na lHQ, constatou-se presença de MP, CP, TLR2 e TLR4 (G1 e G2), maior quantidade de MP (p=0,012) e de TLR4 (p=0,017) no G2. Houve correlação de CP com MP (r=0,810 e p=0,003) e de TLR2 com TLR4 (r=0,569 e p=0,034). Na HIS, constatou-se presença de MP, CP, TLR2 e TLR4 (G1 e G2), não houve diferenças significativas comparando-se os grupos (G1 x G2), porém houve correlação, no G1, de CP com TLR4 (r=0,730 e p=0,040) e de infiltrado inflamatório com células adiposas (r=0,700 e p=0,036). No G2, houve várias correlações: MP com CP (r=0,620 e p=0,016), MP com TLR4 (r=0,662 e p=0,010), CP com TLR2 (r=O,733 e p=0,003), CP com TLR4 (r=0,589 e p=0,027) e de TLR2 com TLR4 (r=0,714 e p=0,004). A PCR em tempo real mostrou presença de CP, pela segunda extração de DNA realizada (G2). Não houve diferença de expressão dos TLRs entre os grupos. A expressão de TLR2 foi maior do que de TLR4 no G1 (p=0,006). O grau de inflamação e o acúmulo de gordura foram maiores no G2 do que no G1(p=0,001). Estes dados sugerem uma relação da co-infecção CP e MP, na gravidade do processo inflamatório presente em placas ateroscleróticas e em pacientes com infarto do miocádio, como também, participação dos receptores Toll-like 2 e 4. / The atherosclerosis is a complex and multifactorial process that is not still completely elucidated. It has been proposed that immune-mediate response to inflammatory and/or infectious processes is implicated in the pathogenesis of the atherosclerotic lesions. Toll-like receptors (TLRs) are involved in the innate response and other physiological events through binding to endogenous and exogenous ligands and it may be involved in the atherogenic process To investigate the Toll-like receptor 2 (TLR2) and Toll-like receptor 4 (TLR4) expression in atheroma plaques and its association with the presence of infectious agents such as Chlamydia pneumoniae (CP) and Mycoplasma pneumoniae (MP) in patients with myocardial infarction (MI) and aortic aneurysms. Fragments of ascending aorta were obtained from MI patients submitted to surgeries of revascularization of the myocardium (G1, n=13) and correction of aortic aneurism (G2, n=14). Frozen and paraffined samples slices were analyzed by Immunohistochemistry (lHQ) and in situ Hybridization for detection and localization of TLR2 and TLR4 expression and CP and MP antigens. There was semiquantification in microscope (0, absent; 1, discreet and focal; 2, moderate and focal; and 3, intense and diffuse). Histopathology was also carried out to investigate the inflammation degree and fat accumulation in these tissues. Real time PCR using SYBR Green System detection was used to stydy DNA CP and MP, also to analyze expression of mRNA TLR2 and TLR4. Using lHQ, it was verified presence of MP, CP, TLR2 and TLR4 (G1 and G2), larger amount of MP (p=0.012) and TLR4 (p=0.017) in G2. In G1 group, MP was positively correlated with CP (r=0.810, p=0.003), in G2, TLR2 with TLR4 (r=0.569, p=0.034). Using HIS, it was verified presence of MP, CP, TLR2 and TLR4 (G1 and G2), there were not significant differences between groups (G1 x G2), however, It was shown correlation between in G1, CP with TLR4 (r=0.730, p=0.040) and also inflammation with fat accumulation (r=0.700, p=0.036). In G2, there were several correlations: presence of MP with CP (r=0.620, p=0.016), MP with TLR4 (r=0.662, p=0.010), CP with TLR2 (r=0.733 p=0,003), CP with TLR4 (r=0.589, p=0.027) and TLR2 with TLR4 (r=0.714, p=0.004). Real time PCR showed presence of CP DNA using second purification accomplished (G2). There was not difference of expression TLRs among the groups. The expression of TLR2 was higher than TLR4 in G1 (p=0.006). Increased degree of inflammation and fat accumulation was also find in G2 than in G1 (p=0.001). These results are suggesting that the gravity of the inflammatory process in atherosclerotic plaques strongly are related to the presence of MP and CP co infection and expression of TLR2 and TLR4, as well in MI patients under myocardial revascularization.

Les infections et l’activation du système immunitaire stimulent l’hématopoïèse. L’activation des récepteurs Toll-like (TLRs) des cellules souches hématopoïétiques, par leur reconnaissance de motifs moléculaires portés par des agents infectieux, en oriente la différenciation vers les voies myéloïdes, renforçant la capacité de notre organisme à lutter contre les infections. Ici, nous avons étudié si les agonistes TLRs peuvent, au contraire, induire au sein de la moelle osseuse l’émergence de progéniteurs hématopoïétiques présentant des propriétés immunorégulatrices. Nous montrons que l’incubation de moelle osseuse de souris en présence de l’agoniste TRL-9, CpG-B, entraîne l’émergence d’une population de progéniteurs au stade pro-B (appelée CpG-proBs). Le transfert adoptif de seulement 60,000 CpG-proBs par receveur, à l’apparition des premiers signes cliniques, confère une protection à long terme dans deux modèles expérimentaux de maladies auto-immunes, le Diabète de Type I (T1D) et l’Encéphalomyélite Auto-immune Expérimentale (EAE). La migration, la différenciation, et les mécanismes cellulaires et moléculaires de cette population protectrice sont décrits et comparés entre ces deux modèles. Dans les deux modèles, les CpG-proBs migrent vers le tissu cible de la réponse auto-immune et se différencient en cellules B matures régulatrices. Dans le T1D, l’interféron-γ (IFN-γ) produit par les cellules T s’avère essentiel pour induire la surexpression de FasL à la surface des CpG-proBs, entraînant l’apoptose des cellules T effectrices. De plus, l’IFN-γ produit par les CpG-proBs réduit la production par les cellules T de l’IL-21, une cytokine pathogène majeure dans le T1D. La descendance des CpG-proBs est composée de précurseurs transitionnels B, de cellules B de la zone marginale et de cellules B folliculaires, exprimant de forts niveaux de FasL et toujours capables d’induire l’apoptose des cellules T, prolongeant ainsi le contrôle des cellules effectrices T auto-immunes in vivo. Dans l’EAE, l’IFNγ est indirectement responsable de la rétention des cellules T, par l’internalisation de CCR7, au sein des ganglions lymphatiques, inhibant ainsi leur migration au système nerveux central (SNC). Dans la moelle épinière, tissu cible de l’EAE, les CpG-proBs se différencient en cellules B220+CD5+CD1dhiCD11b+, secrétant la cytokine anti-inflammatoire IL-10. Enfin, la mobilisation des progéniteurs hématopoïétiques par un cocktail de facteurs hématopoïétiques confère à une sous-population multipotente au stade MPP2 la propriété d’augmenter l’expansion des Foxp3+ Tregs et de prévenir la survenue du diabète de type 1. Nous montrons que les MPP2 mobilisés s’avèrent également capables d’exercer un effet protecteur envers l’EAE. Leur capacité à induire l’expansion de Treg Foxp3+ au sein du SNC et à la périphérie joue un rôle essentiel dans la protection des souris envers l’EAE, puisque la déplétion des Treg abolit la protection déjà établie. Pour conclure, nous avons mis en évidence que diverses stimulations de l’hématopoïèse induisent l’émergence de nouvelles populations de progéniteurs hématopoïétiques qui présentent des propriétés immunorégulatrices et constituent de nouveaux outils de thérapie cellulaire des maladies auto-immunes. / It is well known today that various infectious events or other stimuli of the immune system can trigger hematopoiesis. The hematopoeitic stem and/or progenitor cells express on their cell surface Toll-like receptors which can recognize molecular motifs of infectious agents. The stimulation of TLRs on hematopoietic stem cells favors their differentiation into myeloid lineages, reinforcing the capacity of our body to fight against the pathogens. Herein, we have investigated whether the stimulation of TLRs can induce, instead, the emergence within the bone marrow of selective progenitor cells with immunoregulatory properties. We show that incubation of bone marrow cells with the TLR-9 ligand CpG-B can induce a pro-B cell population (named CpG-proBs) whose adoptive transfer at low numbers of 60,000 cells provided long-lasting protection in two models of autoimmune diseases, Type I Diabetes (TID) and Experimental Autoimmune Encephalomyelitis (EAE) at the onset of clinical signs. The migration, differentiation and molecular mechanism of action of this protective population is described and compared between these two models. In both models, the CpG-proBs migrate to the target tissue of autoimmune responses and differentiate into more mature regulatory B cells. In TID, IFN-γ produced by both T and CpG-proB cells is essential for the upregulation of FasL at the surface of CpG-proBs, inducing the apoptosis of the effector T cells. In addition, IFN-γ reduced the T-cell production of IL-21, a major pathogenic cytokine in TID. The progeny of the adoptively transferred CpG-proBs, including transitional precursors B cells, marginal zone and follicular B cells, display high expression of FasL, promote apoptosis of effector T cells and prolong the control of autoimmune effector T cells in vivo. In EAE, IFN-γ was responsible for the restriction of T cells to the lymph nodes, inhibiting their homing to the CNS. IFN-γ indirectly induced the internalization of CCR7, a receptor required for the migration across the blood-brain barrier. In the spinal cord (target tissue in EAE), CpG-proBs differentiated into B220+CD5+CD1dhiCD11b+ cells secreting the anti-inflammatory cytokine IL-10. Finally, hematopoietic progenitor populations mobilized to the periphery by a cocktail of G-CSF and Flt3l, at the stage of MPP2, have already been shown to protect against TID by expanding the Foxp3+ Tregs. We evaluated them in the EAE model, showing that the ability of these mobilized progenitor cells to trigger the expansion of Foxp3+ Treg within the CNS and the periphery was necessary for providing protection to EAE mice since Treg depletion abrogated the protection once established. In conclusion, we provide evidence for the emergence of new populations of hematopoietic progenitor cells which can display immunoregulatory properties and might be used for cell therapy of autoimmune diseases.